PROJECT SUMMARY The transforming growth factor ? (TGF?) superfamily is the largest family of secreted proteins in mammals. These dimeric ligands, which function in nearly every developmental, physiologic, and pathophysiologic process, including infertility, signal through a heterodimeric complex of type 2 and type 1 serine-threonine kinase receptors that phosphorylate downstream regulatory SMAD proteins and bind SMAD4 to regulate transcription. With NICHD support that started with a physician scientists award (K11HD00960; 1991-93) and this R01 grant (1994-present), we have been productive leaders in the identification and characterization of the oocyte-secreted TGF? family members, growth differentiation factor 9 (GDF9) and bone morphogenetic protein 15 (BMP15), and granulosa cell-secreted activins and inhibins. We have published extensively in this field including >30 papers in Nature, Nature Genetics, Nature Medicine, PLoS Biology, PLoS Genetics, PNAS, and Science. Whereas mammalian oocytes were initially hypothesized to be passengers rather than drivers in ovarian folliculogenesis, we showed that GDF9 is essential for fertility, discovered the X-linked BMP15 gene, and showed that GDF9:BMP heterodimers are the most active oocyte-secreted ligand in mice and women. These insights have defined the oocyte-somatic cell dialogue in ovarian folliculogenesis. In parallel, we showed that inhibin ?-knockout mice are infertile, develop ovarian cancers and die due to an activin-induced cachexia syndrome. BMPs, GDF9:BMP15, activins, and myostatin share common type 2 receptors [activin receptor type 2A (ACVR2A) or type 2B (ACVR2B) or BMPR2], type 1 receptors (ALK4 and ALK5), and receptor-regulated SMADs (SMAD1,2,3,5). Using mouse genetics, we have shown that these proteins function in the pituitary, ovaries, and uterus (e.g., granulosa-specific knockout of SMAD2 and SMAD3 leads to cumulus defects and infertility due to defective GDF9:BMP15 signaling, whereas uterine-specific knockout of SMAD2 and SMAD3 leads to infertility secondary to endometrial hyperplasia). Although SMAD2 and SMAD3 play redundant roles in GDF9:BMP15, activin, and TGF? signaling, we know little about the transcriptional complexes or DNA sequences that they bind. In addition, there are no small molecule inhibitors of ACVR2A/2B and BMPR2. Our overall hypothesis is that oocyte GDF9:BMP15, granulosa cell activins, and uterine BMPs and TGF?s signal through unique SMAD-mediated transcriptional complexes to regulate ovarian and uterine physiology in mice and women. Our proposal will take advantage of state-of-the-art CRISPR/Cas9 strategies to manipulate the mouse genome and DNA-encoded chemical libraries to create novel inhibitors of ACVR2A/2B and BMPR2 and perform follow-up genetic, proteomic, and biochemical approaches to reach our goals. At the end of 5 years, we expect to have unlocked key molecular events that are orchestrated by TGF? family ligands in the female reproductive tract, thereby accelerating translational research to optimize assisted reproductive procedures for women and to create the first ACVR2A/2B and BMPR2 specific small molecules to regulate female fertility.